This invention relates to immunological methods and devices for detecting analytes in biological samples.
Numerous approaches have been developed for detection of a given analyte in a biological sample. Typical of these methods are the so called “lateral flow” and “flow-through” devices and methods. The flow-through device generally uses a porous material with a reagent-containing matrix layered thereon or incorporated therein. Test sample is applied to and flows through the porous material, and analyte in the sample reacts with the reagent(s) to produce a detectable signal on the porous material. These devices are generally encased in a plastic housing or casing with calibrations to aid in the detection of the particular analyte.
Lateral flow assays also utilize a porous membrane for performing analyte detection. Instead of drawing the sample through the membrane perpendicularly, the sample is permitted to flow laterally from an application zone to a reaction zone on the membrane surface. The capture reagent is present in the reaction zone, and the captured analyte can be detected by a variety of protocols, including direct visualization of visible moieties associated with the captured analyte.
One-step lateral flow assays permit a user to add a sample to a sample application region and obtain a positive or negative signal signaling the presence or absence of the test analyte in the sample.
One-step lateral flow devices contain a sample application region to which the liquid sample is applied. The sample application region is in lateral flow contact with the porous carrier material of the analyte detection region. During lateral flow, the sample is brought into contact with a mobile indicator reagent in a discrete zone of the analyte detection region. The indicator reagent contains both a binding moiety which specifically binds to the target analyte and an indicator moiety, which is most often a chromophore label. Target analyte molecules moving in the lateral flow bind to the indicator reagent and are ultimately immobilized in the capture zone, usually by binding to a second reagent which binds specifically to the analyte or to the analyte-indicator reagent complex, giving rise to a positive test signal. Additional signals may include a negative reaction indicator, a test complete indicator, and a positive control indicator.
One-step immunochromatographic devices containing the indicator reagent in a discrete zone of the lateral flow porous material, e.g., at a discrete site on the test strip, have been described.
Lateral flow assays also utilize a porous membrane for performing analyte detection. Instead of drawing the sample through the membrane perpendicularly, however, the sample is permitted to flow laterally from an application zone to a reaction zone on the membrane surface. The capture reagent is present in the reaction zone, and the captured analyte can be detected by a variety of protocols, including direct visualization of visible moieties associated with the captured analyte.
For example, Hochstrasser, U.S. Pat. No. 4,059,407, discloses a dipstick device which can be immersed in a biological fluid to semi-quantitate analyte in the fluid. Semi-quantitation of the analyte is accomplished by using a series of reagent-containing pads wherein each pad in the series will produce a detectable color (i.e., a positive result) in the presence of an increasing amount of analyte.
In U.S. Pat. Nos. 4,094,647, 4,235,601 and 4,361,537, Deutsch et al. describe immunoassays of certain liquid samples deposited on a chromatographic test strip device. The device comprises a material capable of transporting a solution by capillary action, i.e., wicking. Different areas or zones in the strip contain the reagents needed to produce a detectable signal as the analyte is transported to or through such zones. The device is suited for both chemical assays and binding assays which are typified by the binding reaction between an antigen and its complementary antibody.
Many variations on the Deutsch et al. device have also been disclosed. For example, Grubb et al., U.S. Pat. No. 4,168,146 describes the use of a porous test strip material to which is covalently bound an antigen-specific antibody. In performance of an assay, the test strip is immersed in a solution suspected of containing an antigen, and capillary migration of the solution up the test strip is allowed to occur. As the antigen moves up the test strip it binds to the immobilized antigen-specific antibody. The presence of antigen is then determined by wetting the strip with a second antigen-specific antibody to which a fluorescent or enzyme label is covalently bound. Quantitative testing can be achieved by measuring the length of the strip that contains bound antigen.
In addition, European Publication No. 323,605 discloses an assay device using chromatographic material wherein the test sample can travel from one end to the other by capillary action. The chromatographic material contains an immobilized capture reagent capable of binding to the analyte. The application pad receives the test sample and contains a diffusive indicator reagent capable of migrating from the application pad to the chromatographic material. The indicator reagent is capable of binding to the analyte or the capture reagent. The binding of the indicator reagent results in a detectable signal.
Other disclosures of lateral flow assays have also appeared. For example, U.S. Pat. No. 4,861,711 describes a lateral flow assay wherein all components needed for the detection of an analyte are embedded in a single sheet. The lateral flow is referred to as chromatographic behavior. This patent discloses the use of enzyme antibody conjugates and substrates, each separately held in absorbent pads. European Patent Application 306,772 describes a lateral flow device which comprises a chromatographic medium wherein the zone for application of sample and the reaction zone with an immobilized reagent capable of binding the analyte or a label-specific binding material are separated. British Application No. 2,204,398 describes the use of a lateral flow device for testing hCG in urine samples, where sample applied to the device picks up labeled reagent and permeates into a detection zone. Labels include gold sols and colored particles.
For instance, European Publication No. 323,605 discloses an assay device using chromatographic material wherein the test sample can travel from one end to the other by capillary action. The chromatographic material contains an immobilized capture reagent capable of binding to the analyte. The application pad which receives the test sample also contains a diffusible indicator reagent capable of migrating from the application pad to the chromatographic material. The indicator reagent is capable of binding to the analyte. The binding of the indicator reagent-analyte complex results in a detectable signal at the capture situs.
PCT application No. WO 94/06013 also describes a lateral flow assay in which the indicator reagent has been placed in a separate labeling reagent region or pad (referred to as “the third liquid permeable material”). The sample is added to a separate sample application pad, passes through a second permeable material, and mobilizes the indicator reagent located in the third liquid permeable material. The sample then enters the wicking material containing the capture zone. Patent application WO 92/01226 describes a lateral flow device in which the labeled specific binding reagent is retained in the dry state either in a zone on the carrier material or in a separate porous body through which the sample passes en route to the porous carrier material of the test strip.
U.S. patent application Ser. No. 08/444,238 and its corresponding PCT application 96/04748 also describe lateral flow assay devices in which the labeled reagent for the analyte is located in a discrete zone of the porous carrier material of the analyte detection region.
Procedures using chromogenic and fluorescent dyes as labels in biological assay procedures are also known. Typical assay protocols call for direct or indirect binding of a dye label to an analyte or analyte analog in a biological sample, where the presence or absence of the dye at a particular stage of the assay can be determined visually and related to the amount of analyte initially present in the sample. A wide variety of specific assay protocols exist.
A number of those assays utilize naturally colored or dyed particles as a label, where the particles are bound to an antibody or other specific binding substance. Suggested particles include dyed latex beads, dye imbibed liposomes, erythrocytes, metal sols, and the like. The colored particle in such complexes can serve as a visible marker, where separation, capture, or aggregation of the particles is mediated through binding of the antibody or other specific binding substance. The amount of label thus segregated in a particular assay step is related to the amount of analyte initially present in the sample.
U.S. Pat. No. 4,863,875 describes compositions comprising at least ten dye molecules or monomers covalently attached to an antibody through an isocyanate group on the dye. U.S. Pat. No. 4,703,017 describes a solid phase assay device which relies on specific binding of a ligand-label conjugate on a solid support, where the label is disclosed as a particle, such as a liposome, or polymer microcapsule.
For example, U.S. Pat. No. 4,543,522 describes methods of detecting analytes using a solid phase lateral flow assay where the sample is applied to a lateral flow membrane having a pore size of 1-250 microns. PCT Publication WO 92/12428, which is related to the above patent, represents an improvement on that method and device wherein nonbibulous lateral flow is used to conduct visible moieties, especially labeled particles, e.g., dyed latex, red blood cells or liposomes capable of reacting with analyte or a competitor thereto into a capture zone for detection, using a bibulous support made nonbibulous by treatment with a blocking agent. The result is a one-step assay which can be conducted in a very short period of time (typically, within 60 seconds), and wherein the readout is usually available instantaneously upon the sample contacting a capture zone.
Immunoassays have been developed to detect the presence or absence of a variety of analytes including analytes useful in clinical diagnoses, including the diagnosis of Group A Streptococcus. 
Group A Streptococcus is one of the most important causes of acute upper respiratory tract infection. Approximately 19% of all upper respiratory tract infections are caused by Group A Streptococci. (Lauer, B. A., et al., J. Clin. Microb., 17:338-340 (1983)). Early diagnosis and treatment of Group A Streptococcal pharyngitis has been shown to reduce the severity of symptoms and further complications such as rheumatic fever and glomerulonephritis. (Youmans, G. P., et al., in The Biologic and Clinical Basis of Infectious Diseases, at 177-183 (W.B. Saunders Co. 1980)). Conventional identification procedures for Group A Streptococcus from throat swabs involve the isolation and subsequent identification of viable pathogens by techniques that require 24 to 48 hours or longer. (Faklam, R. R. and Washington, J. A., Streptococcus and Related Catalase-Negative Gram-Positive Cocci, in Manual of Clinical Microbiology at 238-257 (Balows, A. et al., eds., 5th ed. 1991)). Immunoassays which detect Group A Streptococcal antigens can be performed in less than one hour.
Various immunoassays for the detection of Group A Streptococcus from throat swabs are commercially available. Immunoassays for Group A Streptococcus sold by Applied Biotech, Inc. and Abbott require the transfer/application of a solubilized sample to the immunoassay device and the addition of three reagents in a specific order. In the Abbott test, the throat swab sample is extracted in a mixture of 3 drops of 2.0 M sodium nitrite and 3 drops of 1.0 M acetic acid which are mixed just prior to the extraction procedure. The swab is introduced into this solution, and twirled to obtain mixing. The sample solution is then neutralized with 3 drops of a solution of 1.0 M Tris buffer prior to running the immunoassay. The immunoassay device contains a plastic housing with a sample well into which the extracted sample is poured.
Similarly, in the Applied Biotech test, sold under the trademark “SURESTEP”, the throat swab sample is extracted in a mixture of 3 drops of 1.0 M sodium nitrite and 3 drops 1.25 M acetic acid which are mixed just prior to the extraction procedure. After the extraction procedure, the sample mixture is neutralized with 3 drops of a solution of 0.1 M Tris-0.7 M sodium hydroxide prior to running the immunoassay. The immunoassay device contains a plastic housing with a sample chamber into which the extracted sample is pipetted.
In addition, U.S. Pat. No. 5,591,645 describes a solid phase chromatographic immunoassay for detecting Group A streptococcus. The assay requires the use of three reagents which must be added in a specific order, i.e., 0.1 M HCl was added to 4M sodium nitrite to obtain nitrous acid. An aliquot of Group A streptococcus was added to the nitrous acid solution. 1 M Tris base was then added to the sample in order to neutralize the nitrous acid prior to running the immunoassay on a dilution series of samples. The end point of the test was 5×105 organisms/ml.
Such tests requiring the addition of more than two reagents introduce the possibility of user error in the sequence of addition of the reagents, necessitating performance of the test by a skilled worker in order to obtain reliable results.
Other tests which do not require transfer of a sample after extraction have complex housings with an area designed for in-the-device sample extractions. The housing of these tests contain an area designed to receive the swab, and requires use of specially designed swabs which fit precisely into the swab chamber area.
The Binax “NOW Strep A test consists of a complex folding booklet cardboard housing which contains an area on the inner right side into which the swab is inserted between layers of cardboard. 4 drops of 2 M sodium nitrite with TWEEN 20 detergent and 4 drops of 0.125 M acetic acid with TWEEN 20 detergent are then added to the swab area, and the swab is rotated. This requires use of a specially designed swab which will fit into the hole designed to receive the swab. The cardboard housing is then folded, bringing the immunoassay strip, housed in the left inner surface, into contact with the swab.
The Quidel “QUICKVUE” strep Test contains a complex plastic housing having a specially designed “in-line” swab chamber into which a specially designed swab is inserted. The “QUICKVUE” strep test contains an extraction solution bottle obtaining 0.6 ml of 4M sodium nitrite with 0.01% Thimerosal, and an internal crushable ampule of 0.65 ml 0.2M acetic acid. The ampule is crushed to mix the solutions just prior to sample extraction. The throat swab specimen is inserted to the swab chamber, and 8 drops of the freshly mixed solution are added to the swab chamber. As the liquid seeps through the swab, the liquid is carried by capillary action into the test strip. Use of a swab which does not precisely fit into the plastic chamber will result in liquid flow which is too rapid for efficient sample extraction.
These one-step assays are complex devices requiring a number of immunoassay reagents. Moreover, because the geometry of these devices for these one-step assays limits the amount of mixing of the sample with the extraction reagents and/or the time of exposure of the sample to the extraction reagents, these assays have limited sensitivity due to poor extraction of the analyte. Thus, there is a need for a one-step assay which permits thorough mixing of the sample with the extraction reagents, and exposure of the sample to the extraction reagents for a desired length of time. Simplifying the number of reagents added during the performance of the assay and eliminating the need to transfer the sample after extraction is also desirable in an assay for health and safety and regulatory purposes. When two reagents are added to the sample during performance of the assay, simplification can also be obtained if the reagents are not required to be added to the sample in a particular sequence.
Thus, there is still the need to develop an immunoassays for a Strep A antigen extracted or solubilized from samples, where 2 or less sample extraction reagents are added, in no particular sequence, during performance of the assay, and where the sample does not require transfer to the immunoassay devise after efficient sample extraction. There is also a need for a one-step assay utilizing devices which do not require complex plastic or cardboard housings or specially designed swabs to obtain sample extraction.
None of the references or products described herein is admitted to be prior art.